We have previously studied oral tolerance to myelin basic protein (MBP) in the Lewis rat model of EAE. We have demonstrated that feeding MBP generates regulatory cells that suppress EAE by the release of TGF-beta after being triggered by the fed antigen. More recently, we have found that orally administered antigen also generates T cells that are of the Th2 type in that they secrete IL-4 after being triggered by the fed antigen. We have also found that depending on the dose of antigen fed, active suppression or clonal anergy is generated and can be demonstrated according to cytokine patterns. We have now initiated studies of oral tolerance in murine models of EAE which will allow better immunologic characterization of the mechanisms of oral tolerance. The following specific aim will be addressed: 1) In MBP-TcR transgenic mice, to what degree does active suppression, clonal deletion and clonal anergy occur following orally administered antigen? Three potential mechanisms of antigen driven peripheral tolerance can result following orally administered antigen. Investigations using MBP-TcR transgenic animals will allow a delineation of which of these pathways occur and the factors that influence one pathway over the other. We hypothesize that low dose oral antigen is processed through a unique mucosal pathway to activate regulatory T cells in Peyer's patches whereas high dose oral antigen leads to clonal anergy via systemic delivery of antigen following passage through the gut. The MBP-TcR animals will also allow us to determine to what degree clonal deletion occurs following oral antigen and whether anergy occurs locally in the gut in association with oral tolerance. 2) What are the relative roles of TGF-beta, IL-10 and IL-4 in mediating oral tolerance? Orally administered antigens generate regulatory cells which act by secreting suppressive cytokines following antigen specific triggering. The relative role of these cytokines in oral tolerance will be investigated using anti-cytokine antibodies administered in vivo, cytokine transgenic animals, and cytokine deficient animals. 3) What are the roles of costimulatory molecules in the induction of oral tolerance? A fundamental question related to oral tolerance is why orally administered antigen preferentially activates cells producing IL-4, IL-10 and TGF-beta. We hypothesize that T cells producing these cytokines require costimulatory signals qualitatively different from those required for Th1 cells and that the mucosal pathway of antigen presentation provides signals for activating Th2 and TGF-beta T cells, but anergize Th1 cells. This will be investigated using monoclonal antibodies to B7 and other costimulatory molecules and in B7 deficient mice. In summary, the studies outlined will help provide a basic understanding of the mechanisms of oral tolerance in murine experimental models.